Vascular endothelial cells and blood platelets play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the bloodstream. For example, endothelial cells preferentially bind monocytes and granulocytes prior to their migration through the blood vessel wall and into surrounding tissue in an inflammatory response.
Certain inflammation-triggering compounds are known to act directly on the vascular endothelium to promote the adhesion of leukocytes to vessel walls. Cells then move through the walls and into areas of injury or infection.
Intercellular adhesion to vascular endothelium is also thought to be involved in tumor metastasis. Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.
Blood platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane fuses with the external plasma membrane. As a result, new cell surface proteins become expressed that confer on the activated platelet new functions, such as the ability to bind both other activated platelets and other cells. Activated platelets are recruited into growing thrombi, or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes that are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.
Recent work has revealed that specialized cell surface receptors on endothelial cells and platelets, designated endothelial leukocyte adhesion molecule-1 (ELAM-1) and granule membrane protein-140 (GMP-140), respectively, are involved in the recognition of various circulating cells by the endothelium and platelets. For example, ELAM-1 has been shown to mediate endothelial leukocyte adhesion, which is the first step in many inflammatory responses. Specifically, ELAM-1 binds human neutrophils, monocytes, eosinophils, certain T-lymphocytes [Graber et al., J. Immunol., 145:819 (1990)], NK cells, and the promyelocytic cell line HL-60.
ELAM-1 is inducibly expressed on vascular endothelial cells [Bevilacqua et al., Science, 243:1160-1165 (1989) and Hession et al., Proc. Natl. Acad. Sci., 87:1673-1677 (1990)]. This receptor has been demonstrated to be induced by inflammatory cytokines such as interleukin I.beta. (IL-I.beta.) and tumor necrosis factor .alpha. (TNF.alpha.), as well as bacterial endotoxin (lipopolysaccharide) [Bevilacqua et al., Proc. Natl. Acad. Sci., 84:9238-9242 (1987)]. These compounds augment polymorphonuclear leukocyte (neutrophil), and monocyte adhesion [Bevilacqua et al., Proc. Natl. Acad. Sci., 84:9238-9242 (1987)].
GMP-140 (also known as PADGEM) is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions, [Geng et al., Nature, 343:757-760 (1990)]. Thus, for example, activated platelets that express GMP-140 on their surfaces are known to bind to monocytes and neutrophils [Jungi et al., Blood,m 67:629-636 (1986)], and also to bind monocyte-like cell lines, e.g., HL60 and U937 [Jungi et al., Blood, 67:629-636 (1986); Silverstein et al., J. Clin. Invest., 79:867-874 (1987)].
GMP-140 is an alpha granule membrane protein of molecular mass 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion [Hsu-Lin et al., J. Clin. Chem., 259:9121-9126 (1984); Stenberg et al., J. Cell Biol., 101:880-886 (1985); Berman et al., J. Clin. Invest., 78:130-137 (1986)]. It is also found in megakaryocytes [Beckstead et al., Blood, 67:285-293 (1986)], and in endothelial cells [McEver et al., Blood, 70:335a (1987)]within the Weibel-Palade bodies [Bonfanti et al., Blood, 73:1109-1112 (1989)]. Furie et al., U.S. Pat. No. 4,783,330, describe monoclonal antibodies reactive with GPM-140.
A third receptor is the lymphocyte homing receptor, MEL-14 antigen or LAM-1 [Gallatin et al., Nature, 304:30-34 (1983); Siegellman et al., Science, 243:1165-1172 (1989); Rosen, Cell Biology, 1:913-919 (1989); and Lasky et al., Cell, 56:1045-1055 (1989)]. In addition to lymphocyte homing, MEL-14 antigen/LAM-1 is believed to function early in neutrophil binding to the endothelium.
The term "selectin" has been suggested for a general class of receptors, which includes ELAM-1 and GMP-140 and MEL-14, because of their lectin-like domain and the selective nature of their adhesive functions. The structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors [Bevilacqua et al., Science, 243:1160-1165 (1989), (ELAM-1); Geng et al., Nature, 343:757-760 (1990), (GMP-140); and Lasky et al., Cell, 56:1045-1055 (1989), (MEL-14 antigen)].
The extracellular portion of selectins can be divided into three segments based on homologies to previously described proteins. The N-terminal region (about 120 amino acids) is related to the C-type mammalian lectin protein family as described by Drickamer, J. Biol. Chem., 263:9557-9560 (1988) that includes low affinity IgE receptor CD23. A polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif. Lastly, after the EGF domain are one or more tandem repetitive motifs of about 60 amino acids each, related to those found in a family of complement regulatory proteins.
The above-described selectins are now referred to as E-, P- and L-selectins that correspond to ELAM-1, GMP-140 and MEL-14 antigen/LAM-1, respectively. Cell adhesion of leukocytes to endothelial cells involves the interaction of E-, P- and L-selectins with their respective receptors. [Paulson, J. C. in "Adhesion, Its role in Inflammatory Disease", (Harlan, J.; Liu, D. eds.) W. H. Freeman, New York, Chapt. 2, p.19 (1992); Springer et al. Nature, 349:196 (1991); Lasky, Science, 258:964 (1992); Kobata et al. in "Cell Surface Carbohydrates and Cell Development", (Fukuda, M., Ed.), CRC Press, London, p.1 (1992)]. Although the natural ligands have not been completely characterized, the partial chemical structure of the ligands for E- and P-selectin has been shown to contain the tetrasaccharide sialyl Lewis X (SLe.sup.x) [Phillips et al., Science, 250:1130 (1990); Walz et al., Science, 250:1132 (1990); Lowe et al., Cell, 63:475 (1990); Polley et al., Proc. Natl. Acad. Sci. USA, 88:6224 (1991); Zhou et al., J. Cell Biol., 88:557 (1991); U.S. Pat. Nos. 5,079,353 and 5,296,594], although sialyl Lewis A type structures may also act as receptor ligands. [Berg et al., Biochem Biophys. Res. Commun., 184:1048 (1992); Berg et al., J. Biol Chem., 23:14869 (1991); Handa et al., Biochem. Biophys. Res. Commun., 181:1223 (1991)]. Sialyl Le.sup.x glycal has also been shown to inhibit binding to E-selectin. [DeFrees et al., J. Am. Chem. Soc., 115:7549 (1993).]
The ligand for L-selectin has also been proposed to contain an SLe.sup.x type structure in which the sialic acid is replaced with a sulfate group. [Yuen et al., Biochemistry, 31:9126 (1992)]. Application WO 92/22564 discloses sulfate, phosphate and carboxylate derivatives of Lewis X and Lewis a compounds that lack a sialyl group, but are said to provide enhanced immunosuppressing or tolerogenic properties over derivatives lacking the sulfate, phosphate or carboxylate substituents.
Published International application WO 91/19501 and WO 91/19502 disclose that oligosaccharides containing the pentameric and hexameric structures shown below inhibited selective intercellular binding between cells containing the ligand (below) and those containing a selectin receptor and that the penta- and hexasaccharides provided better inhibition than did SLe.sup.x :
NeuAc.alpha.2.fwdarw.3.beta.Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc .beta.1,3Gal.beta.-; PA1 NeuAc.alpha.2.fwdarw.3.beta.Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc .beta.1,3Gal.beta.1,4Glc-; and PA1 NeuAc.alpha.2.fwdarw.3.beta.Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc =SLe.sup.x. PA1 R.sup.2 is selected from the group consisting of a C.sub.1 -C.sub.18 aliphatic, an aryl, a substituted aryl and a phenyl C.sub.1 -C.sub.3 alkylene, wherein an aryl group has one six-membered aromatic ring or two fused six-membered aromatic rings, which ring or rings are hydrocarbyl, monoazahydrocarbyl, or diazahydrocarbyl rings, and a substituted aryl group is a before-mentioned aryl group having a substituent selected from the group consisting of halo, trifluoromethyl, nitro, C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy, amino, mono-C.sub.1 -C.sub.6 alkylamino, di-C.sub.1 -C.sub.6 alkylamino, benzylamino and C.sub.1 -C.sub.6 alkylbenzylamino; PA1 R.sup.3 is methyl or hydroxymethyl; PA1 X is selected from the group consisting of hydroxyl, C.sub.1 -C.sub.6 acyloxy, C.sub.1 -C.sub.6 hydroxylacyloxy, halo and azido; PA1 Z.sup.1 and Z.sup.2 are .alpha.-L-fucosyl or hydrogen (H), but at least one of Z.sup.1 and Z.sup.2 is .alpha.-L-fucosyl; and PA1 M is a proton (H.sup.+) or a pharmaceutically acceptable cation.
Mulligan et al., Nature, 364:148 (1993) showed that infusion of SLe.sup.x or a SLe.sup.x -galactoside that are ligands for P-selectin reduced lung injury and reduced accumulation of neutrophils in cobra venum-infused rats. U.S. Pat. No. 5,143,712 and application WO 92/22301 similarly and separately disclose that LacNAc linear multimers having a non-reducing terminal SLe.sup.x or sialyl-LacNAc group can be useful in suppressing an immune response.
Free oligosaccharides, both natural and synthetic, generally exhibit weak binding to lectin receptors, a deficiency that is often overcome by multivalent interactions. [Kingery-Wood et al., Am. Chem. Soc., 114:7303 (1992); Weis et al., Nature, 360:127 (1992); Sabesan et al., J. Am. Chem. Soc., 114:8363 (1992)]. The fact that monomeric SLe.sup.x binds weakly to E- and P-selectin, [Nelson et al., J. Clin. Invest., 91:1157 (1993)], coupled with recent observations [Moore et al., J. Cell Biol., 118:445 (1992] that suggest a clustering of SLe.sup.x type structures on the putative glycoprotein ligands reinforces the concept that SLe.sup.x -selectin interactions are multimeric in vivo. Sialyl Lewis X structures on the natural selectin ligand could exist as either N- or 0-linked oligosaccharides, [Kobata et al,, in "Cell Surface Carbohydrates and Cell Development", (Fukuda, M., Ed.), CRC Press: London, p.1 (1992)] and multiple copies of SLe.sup.x may be presented as clusters of single SLe.sup.x units in close proximity or as multiple SLe.sup.x structures on polyantennary oligosaccharide chains.
The present invention, discussed hereinafter, illustrates the capacity of a branched oligosaccharide containing two copies of the SLe.sup.x glycotope to inhibit binding between cells containing an E-selectin receptor and cells that express an E-selectin ligand, such as activated endothelial cells and neutrophils and contemplates several bivalent SLe.sup.x inhibitor analogs that can mimic structures found in N- and O-glycans.